Compositions and Methods for Detection of Antibodies Specific for Anaplasma phagocytophilum (Aph) and Anaplasma platys (Apl)

ABSTRACT

The invention provides methods and compositions for the detection and treatment of  Anaplasma phagocytophilum  and  Anaplasma platys  infection.

PRIORITY

This application is a divisional application of U.S. Ser. No.14/069,489, filed on Nov. 1, 2013, which is a divisional application ofU.S. Ser. No. 13/668,597, filed on Nov. 5, 2012, now U.S. Pat. No.8,580,272, which is divisional application of U.S. Ser. No. 12/575,531,filed Oct. 8, 2009, now U.S. Pat. No. 8,303,959, which claims thebenefit of U.S. application 61/103,743, filed Oct. 8, 2008. Theseapplications are incorporated herein by reference in their entirety.

SEQUENCE LISTING

This document incorporates by reference an electronic sequence listingtext file, which was electronically submitted along with this document.The text file is named 08_(—)1241_SeqListing_ST25_(—)2.txt, is 12,408bytes, and was created on Oct. 5, 2009.

BACKGROUND OF THE INVENTION

Anaplasmosis occurs in mammals, including, e.g., humans, horses, sheep,dogs, cats, deer, and ruminants and is caused by infection ofgranulocytic cells with the tick-borne agent Anaplasma phagocytophilum(“Aph”) (formerly known as Ehrlichia equi). Common clinical symptomsinclude fever, lethargy, lameness, thrombocytopenia, swelling of thelymph nodes, and anexoria, all of which are non-specific toanaplasmosis. Therefore a specific test is important for correctdiagnosis.

Anaplasma platys (“Apl”) (formerly known as Ehrlichia platys) is a veryclosely related species. Apl can cause infectious canine cyclicthrombocytopenia (ICCT). Infected dogs are usually asymptomatic in theU.S.A, but may become clinically ill in other parts of the world.Current serologic tests for Anaplasma can not differentiate the twospecies because of significant cross-reactivity. Methods of detectingAph and Apl and methods of differentiating between the two infectionsare needed in the art.

The onset of clinical symptoms occurs during the acute phase ofanaplasmosis can precede the advent of measurable levels of antibodiesagainst some Anaplasma antigens. Thus, there is a need for a rapid,sensitive and reliable immunological test for Aph infection, Aplinfection, or both, e.g., in mammals exhibiting clinical symptoms ofacute anaplasmosis.

SUMMARY OF THE INVENTION

One embodiment of the invention provides a composition comprising one ormore purified polypeptides consisting of an amino acid sequence setforth as SEQ ID NO:8 or SEQ ID NO:10; or consisting of: amino acids 1 to45 of SEQ ID NO:10; amino acids 41 to 89 of SEQ ID NO:10; amino acids 85to 130 of SEQ ID NO:10; amino acids 126 to 160 of SEQ ID NO:10; aminoacids 129 to 146 of SEQ ID NO:10; amino acids 144 to 160 of SEQ IDNO:10; or amino acids 136 to 155 of SEQ ID NO:10; or consisting of atleast 17 contiguous amino acids of an amino acid sequence set forth asSEQ ID NOs:1-21; or consisting of a polypeptide having at least about94% identity to SEQ ID NOs:1-21. The one or more purified polypeptidescan be linked to an indicator reagent, a signal sequence, a stoptransfer sequence, an amino acid spacer, a transmembrane domain, aprotein purification ligand, a heterologous polypeptide, a moiety thatenhances an immune response, a moiety that facilitates purification, amoiety that facilitates polypeptide stability, one or more additionalpolypeptides comprising SEQ ID NOs:1-21 or a combination thereof.Another embodiment of the invention provides an isolated polynucleotidethat encodes the one or more of the purified polypeptides.

Yet another embodiment of the invention provides a method of detectingantibodies that specifically bind an Anaplasma phagocytophilumpolypeptide or an Anaplasma platys polypeptide or both in a test sample.The method comprises contacting the composition comprising one or morepurified polypeptides with the test sample, under conditions that allowpolypeptide/antibody complexes to form. The polypeptide/antibodycomplexes are detected. The detection of the polypeptide/antibodycomplexes is an indication that antibodies specific for an Anaplasmaphagocytophilum polypeptide or an Anaplasma platys polypeptide or bothare present in the test sample. The composition comprising one of morepurified polypeptides can consist of at least 17 contiguous amino acidsof an amino acid sequence set forth as SEQ ID NOs:8 or 19. The detectionof the polypeptide/antibody complexes is an indication that antibodiesspecific for Anaplasma platys are present in the test sample. Thecomposition comprising one of more purified polypeptides can consist ofat least 17 contiguous amino acids of an amino acid sequence set forthas SEQ ID NO:10; amino acids 41-89 of SEQ ID NO:10; or amino acids85-130 of SEQ ID NO:10; or can consist of a polypeptide having at leastabout 94% identity to SEQ ID NOs:10, amino acids 41-89 of SEQ ID NO:10,or amino acids 85-130 of SEQ ID NO:10. The detection of thepolypeptide/antibody complexes is an indication that antibodies specificfor an Anaplasma phagocytophilum polypeptide or an Anaplasma platyspolypeptide or both are present in the test sample. The compositioncomprising one of more purified polypeptides can consist of at least 17contiguous amino acids of amino acids 1-45 of SEQ ID NO:10; amino acids126-160 of SEQ ID NO:10; amino acids 129-146 of SEQ ID NO:10; aminoacids 144-160 of SEQ ID NO:10; amino acids 136-155 of SEQ ID NO:10; orcan consist of a polypeptide having at least about 94% identity to aminoacids 1-45 of SEQ ID NO:10; amino acids 126-160 of SEQ ID NO:10; aminoacids 129-146 of SEQ ID NO:10; amino acids 144-160 of SEQ ID NO:10;amino acids 136-155 of SEQ ID NO:10. The detection of thepolypeptide/antibody complexes is an indication that antibodies specificfor an Anaplasma phagocytophilum polypeptide are present in the testsample. The complexes can be contacted with an indicator reagent priorto the detection. The amount of antibodies in the test sample can bedetermined. The one or more purified polypeptides can be attached to asubstrate.

Still another embodiment of the invention provides an antibody thatspecifically binds to a polypeptide consisting of SEQ ID NOs:1-21. Theantibody can be a monoclonal antibody, polyclonal antibody, a Fabfragment, a Fab′ fragment, Fab′-SH fragment, F(ab′)₂ fragment, Fvfragment, or a single chain antibody.

Even another embodiment of the invention provides a method of detectingan Anaplasma phagocytophilum polypeptide or an Anaplasma platyspolypeptide or both in a sample. The method comprises contacting one ormore antibodies that specifically bind to the one or more purifiedpolypeptides of the invention with the sample under conditions thatallow polypeptide/antibody complexes to form. The polypeptide/antibodycomplexes are detected. The detection of the polypeptide/antibodycomplexes is an indication that an Anaplasma phagocytophilum polypeptideor an Anaplasma platys polypeptide or both is present in the sample. Theone or more purified polypeptides can consist of at least 17 contiguousamino acids of amino acids 1-45 of SEQ ID NO:10, amino acids 126-160 ofSEQ ID NO:10; amino acids 129-146 of SEQ ID NO:10; amino acids 144-160of SEQ ID NO:10; or amino acids 136-155 of SEQ ID NO:10. The detectionof the polypeptide/antibody complexes is an indication that Anaplasmaphagocytophilum polypeptides are present in the test sample. The one ormore purified polypeptides can consist of at least 17 contiguous aminoacids of an amino acid sequence set forth as SEQ ID NOs:8 or 19. Thedetection of the polypeptide/antibody complexes is an indication thatAnaplasma platys polypeptides are present in the test sample. The one ormore purified polypeptides can consist of at least 17 contiguous aminoacids of an amino acid sequence set forth as SEQ ID NOs:10; amino acids41-89 of SEQ ID NO:10; or amino acids 85-130 of SEQ ID NO:10. Thedetection of the polypeptide/antibody complexes is an indication that anAnaplasma phagocytophilum polypeptide or an Anaplasma platys polypeptideis present in the test sample. The one or more antibodies can bemonoclonal antibodies, polyclonal antibodies, Fab fragments, Fab′fragments, Fab′-SH fragments, F(ab′)₂ fragments, Fv fragments, or singlechain antibodies.

In another embodiment of the invention, a composition comprisingpurified polypeptides of the invention further comprises apharmaceutically-acceptable or veterinarily acceptable carrier and/or anadjuvant.

Yet another embodiment of the invention provides a method of treating orameliorating Anaplasma platys infection, Anaplasma phagocytophiluminfection, or both, in a mammalian subject comprising administering tothe mammalian subject a therapeutically effective amount of acomposition comprising one or more of the purified polypeptides of theinvention.

Even another embodiment of the invention provides a method of inducingan immune response in a mammal comprising administering animmunologically effective amount of a composition comprising one or moreof the purified polypeptides of the invention.

The invention therefore provides methods and compositions for thedetection and treatment of Apl and/or Aph infection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of assays completed with polypeptidescomprising SEQ ID NOs:12-18 and 10.

FIG. 2 shows the results of assays completed with polypeptidescomprising SEQ ID NOs:15, 19, and 10.

FIG. 3 shows the results of assays completed with polypeptidescomprising SEQ ID NOs:15, 19, and 10.

FIG. 4 shows the results of time course assays completed withpolypeptides comprising SEQ ID NOs:15, 19, and 10.

FIG. 5 shows the results of time course assays completed withpolypeptides comprising SEQ ID NOs:15 and 10.

FIG. 6 shows the results of assays completed with polypeptidescomprising SEQ ID NOs:12-15.

FIG. 7A-B shows the results of assays completed with polypeptidescomprising SEQ ID NO:20.

DETAILED DESCRIPTION OF THE INVENTION Polypeptides of the Invention

As used herein, the singular forms “a,” “an”, and “the” include pluralreferents unless the context clearly dictates otherwise.

A polypeptide is a polymer of two or more amino acids covalently linkedby amide bonds. A polypeptide can be post-translationally modified. Apurified polypeptide is a polypeptide preparation that is substantiallyfree of cellular material, other types of polypeptides, chemicalprecursors, chemicals used in synthesis of the polypeptide, orcombinations thereof. A polypeptide preparation that is substantiallyfree of cellular material, culture medium, chemical precursors,chemicals used in synthesis of the polypeptide, etc., has less thanabout 30%, 20%, 10%, 5%, 1% or more of other polypeptides, culturemedium, chemical precursors, and/or other chemicals used in synthesis.Therefore, a purified polypeptide is about 70%, 80%, 90%, 95%, 99% ormore pure. A purified polypeptide does not include unpurified orsemi-purified cell extracts or mixtures of polypeptides that are lessthan 70% pure.

The term “polypeptides” can refer to one or more of one type ofpolypeptide (a set of polypeptides). “Polypeptides” can also refer tomixtures of two or more different types of polypeptides (a mixture ofpolypeptides). The terms “polypeptides” or “polypeptide” can each alsomean “one or more polypeptides.”

One embodiment of the invention provides one or more of the followingpolypeptides:

Aph p44-1 (SEQ ID NO: 1)GSDVRAHDDVSALETGGAGYFYVGLDYSPAFSKIRDFSIRESNGE 45 Aph p44-2(SEQ ID NO: 2) ESNGETKAVYPYLKDGKSVKLESHKFDWNTPDPRIGFKDNMLVAMEG SV 49Aph p44-3 (SEQ ID NO: 3)MEGSVGYGIGGARVELEIGYERFKTKGIRDSGSKEDEADTVYLLAK 46 Aph p44-4(SEQ ID NO: 4) YLLAKELAYDVVTGQTDNLAAALAKTSGKDIVQFA 35 Aph p44-4-1 (SEQ ID NO: 5) AKELAYDVVTGQTDNLAA 18 Aph p44-4-2  (SEQ ID NO: 6)LAAALAKTSGKDIVQFA 17 Aph p44-4-3  (SEQ ID NO: 7) VVTGQTDNLAAALAKTSGKD 20Apl p44-4  (SEQ ID NO: 8) AKKLPHTLVSDQSDKFLEELKNTKAAEIVKFA 32 p44-4-V(SEQ ID NO: 9) YLLAKELAYDVVTGQTDKLTAALAKTSGKDFVQFA 35 Aph rp44(SEQ ID NO: 10) GSDVRAHDDVSALETGGAGYFYVGLDYSPAFSKIRDFSIRESNGETKAVYPYLKDGKSVKLESHKFDWNTPDPRIGFKDNMLVAMEGSVGYGIGGARVELEIGYERFKTKGIRDSGSKEDEADTVYLLAKELAYDVVTGQTDXLXAALAKTSGKDXVQFANAVKISSPTIDGKVCSGDHAAIVSTKGKDYKADPKE SGNNGHETSQCSGLSSS 213In one embodiment of the invention, the X at position 143 of SEQ IDNO:10 is K or N; the X at position 145 is T or A; and the X at position156 is F or I.

One embodiment of the invention provides the following polypeptides:

1. Amino acids 1-45 of SEQ ID NO:10 (Aph p44-1) (which has the samereactivities of SEQ ID NOs:1 and 12).2. Amino acids 41-89 of SEQ ID NO:10 (Aph p44-2) (which has the samereactivities of SEQ ID NOs:2 and 13).3. Amino acids 85-130 of SEQ ID NO:10 (Aph p44-3) (which has the samereactivities of SEQ ID NOs:3 and 14).4. Amino acids 126-160 of SEQ ID NO:10 (Aph p44-4) (which has the samereactivities of SEQ ID NOs:4, 9, 11, 15, and 20).5. Amino acids 129-146 of SEQ ID NO:10 (Aph p44-4-1) (which has the samereactivities of SEQ ID NOs:5 and 16).6. Amino acids 144-160 of SEQ ID NO:10 (Aph p44-4-2) (which has the samereactivities of SEQ ID NOs:6 and 17).7. Amino acids 136-155 of SEQ ID NO:10 (Aph p44-3) (which has the samereactivities of SEQ ID NOs:7 and 18).Furthermore, SEQ ID NOs:8 and 9 have the same reactivities, that is, thepolypeptides specifically bind antibodies specific for Anaplasmaantigens in substantially the same manner.

In one embodiment of the invention, the polypeptides have an N-terminalC-residue. For example:

Aph p44-1 (SEQ ID NO: 12) CGSDVRAHDDVSALETGGAGYFYVGLDYSPAFSKIRDFSIRESNGEAph p44-2 (SEQ ID NO: 13)CESNGETKAVYPYLKDGKSVKLESHKFDWNTPDPRIGFKDNMLVAMEGSV Aph p44-3(SEQ ID NO: 14) CMEGSVGYGIGGARVELEIGYERFKTKGIRDSGSKEDEADTVYLLAKAph p44-4 (SEQ ID NO: 15) CYLLAKELAYDVVTGQTDNLAAALAKTSGKDIVQFAAph p44-4-1 (SEQ ID NO: 16) CAKELAYDVVTGQTDNLAA Aph p44-4-2(SEQ ID NO: 17) CLAAALAKTSGKDIVQFA Aph p44-4-3 (SEQ ID NO: 18)CVVTGQTDNLAAALAKTSGKD Apl p44-4 (SEQ ID NO: 19)CAKKLPHTLVSDQSDKFLEELKNTKAAEIVKFA p44-4-V (SEQ ID NO: 20)CYLLAKELAYDVVTGQTDKLTAALAKTSGKDFVQFA Aph rp44 (SEQ ID NO: 21)CGSDVRAHDDVSALETGGAGYFYVGLDYSPAFSKIRDFSIRESNGETKAVYPYLKDGKSVKLESHKFDWNTPDPRIGFKDNMLVAMEGSVGYGIGGARVELEIGYERFKTKGIRDSGSKEDEADTVYLLAKELAYDVVTGQTDXLXAALAKTSGKDXVQFANAVKISSPTIDGKVCSGDHAAIVSTKGKDYKADPKESGN NGHETSQCSGLSSS.In one embodiment of the invention, the X at position 144 of SEQ IDNO:21 is K or N; the X at position 146 is T or A; and the X at position157 is F or I.

SEQ ID NOs:4-7 and 9 can be aligned as follows:

Aph p44-4 (SEQ ID NO: 4) YLLAKELAYDVVTGQTDNLAAALAKTSGKDIVQFA Aph p44-4-1(SEQ ID NO: 5) AKELAYDVVTGQTDNLAA Aph p44-4-2 (SEQ ID NO: 6)LAAALAKTSGKDIVQFA Aph p44-4-3 (SEQ ID NO: 7) VVTGQTDNLAAALAKTSGKDAph p44-4-v (SEQ ID NO: 9) YLLAKELAYDVVTGQTDKLTAALAKTSGKDFVQFA

A consensus sequence of SEQ ID NOs:4-7 and 9 is shown in SEQ ID NO:11:

SEQ ID NO: 11 YLLAKELAYDVVTGQTDXLXAALAKTSGKDXVQFAIn one embodiment of the invention, the X at position 18 of SEQ ID NO:11is N or K; the X at position 20 is T or A; and the X at position 31 is For I. One embodiment of the invention comprises amino acids 4-21 of SEQID NO:11; amino acids 19-34 of SEQ ID NO:11; or amino acids 11-30 of SEQID NO:11.

One embodiment provides a purified polypeptide that consists of lessthan about 46, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, or 6 contiguous amino acids (or any range between about 46and about 6 amino acids) of SEQ ID NOs:1-9 and 11-21. One embodimentprovides a purified polypeptide that consists of more than about 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, or 46contiguous amino acids (or any range between about 46 and about 6 aminoacids) of SEQ ID NOs:1-9 and 11-12. Naturally occurring Aph or Apl aminoacids are any polypeptides naturally produced by an Aph or Apl organism.A purified polypeptide can comprise less than a certain number ofcontiguous naturally occurring Anaplasma amino acids (e.g., about lessthan 200, 175, 150, 125, 100, 75, 50, 45, 40, 35, 30, 25, 20, 19, 18,17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6 amino acids (or any rangebetween about 200 and about 6 amino acids)) of SEQ ID NOs:1-21. That is,the purified polypeptide is smaller than the full length polypeptide.The fact that these polypeptides are smaller than a full lengthAnaplasma polypeptide is important because smaller polypeptides can havegreater specificity and/or sensitivity than full length polypeptides inApl and/or Aph assays. Additionally, these smaller polypeptides can beless expensive to manufacture, and may be obtained at greater purity,than the full length polypeptide.

Another embodiment provides a purified polypeptide that consists of lessthan about 200, 175, 150, 125, 100, 75, 50, 25, 20, 15, 10, 6 or lesscontiguous naturally occurring Anaplasma amino acids (or any rangebetween about 200 and about 6 amino acids) of SEQ ID NO:10. Anotherembodiment provides a purified polypeptide that consists of more thanabout 6, 10, 15, 25, 50, 75, 100, 125, 150, 175, 200 or more contiguousnaturally occurring Anaplasma amino acids (or any range between about 6and about 200 amino acids) of SEQ ID NO:10.

Variant polypeptides that are at least about 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to thepolypeptide sequences shown in SEQ ID NOs:1-21 are also polypeptides ofthe invention. For example, a variant polypeptide of SEQ ID NO:1 can beabout at least about 98% (about 1 amino acid change), 96% (about 2 aminoacid changes), 93% (about 3 amino acid changes), 91% (about 4 amino acidchanges), 89% (about 5 amino acid changes), 87% (about 6 amino acidchanges), 84% (about 7 amino acid changes), 82% (about 8 amino acidchanges), or 80% (about 9 amino acid changes) identical to SEQ ID NO:1.A variant polypeptide of SEQ ID NO:2 can be about at least 98% (about 1amino acid change), 96% (about 2 amino acid changes), 94% (about 3 aminoacid changes), 92% (about 4 amino acid changes), 90% (about 5 amino acidchanges), 88% (about 6 amino acid changes), about 86% (about 7 aminoacid changes), 84% (about 8 amino acid changes), 82% (about 9 amino acidchanges), or 80% (about 10 amino acid changes) identical to SEQ ID NO:2.A variant polypeptide of SEQ ID NO:3 can be at least about 98% (about 1amino acid change), 96% (about 2 amino acid changes), 94% (about 3 aminoacid changes), 91% (about 4 amino acid changes), 89% (about 5 amino acidchanges), 87% (about 6 amino acid changes), 85% (about 7 amino acidchanges), 83% (about 8 amino acid changes), or 80% (about 9 amino acidchanges) identical to SEQ ID NO:3. A variant polypeptide of SEQ ID NOs:4and 9 can be about at least 97% (about 1 amino acid change), 94% (about2 amino acid changes), 91% (about 3 amino acid changes), 89% (about 4amino acid changes), 86% (about 5 amino acid changes), 83% (about 6amino acid changes), or 80% (about 7 amino acid changes) identical toSEQ ID NOs:4 and 9. A variant polypeptide of SEQ ID NO:5 can be at leastabout 94% (about 1 amino acid change), 89% (about 2 amino acid changes),or 83% (about 3 amino acid changes) identical to SEQ ID NO:5. A variantpolypeptide of SEQ ID NO:6 can be at least about 94% (about 1 amino acidchange), 88% (about 2 amino acid changes), or 82% (about 3 amino acidchanges) identical to SEQ ID NO:6. A variant polypeptide of SEQ ID NO:7can be about at least 95% (about 1 amino acid change), 90% (about 2amino acid changes), 85% (about 3 amino acid changes) or 80% (about 4amino acid changes) identical to SEQ ID NO:7. A variant polypeptide ofSEQ ID NO:8 can be about at least 97% (about 1 amino acid change), 94%(about 2 amino acid changes), 91% (about 3 amino acid changes), 88%(about 4 amino acid changes), 84% (about 5 amino acid changes), or 81%(about 6 amino acid changes) identical to SEQ ID NO:8.

Variant polypeptides have one or more conservative amino acid variationsor other minor modifications and retain biological activity, i.e., arebiologically functional equivalents. A biologically active equivalenthas substantially equivalent function when compared to the correspondingwild-type polypeptide. In one embodiment of the invention a polypeptidehas about 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, or less conservativeamino acid substitutions.

Percent sequence identity has an art recognized meaning and there are anumber of methods to measure identity between two polypeptide orpolynucleotide sequences. See, e.g., Lesk, Ed., Computational MolecularBiology, Oxford University Press, New York, (1988); Smith, Ed.,Biocomputing: Informatics And Genome Projects, Academic Press, New York,(1993); Griffin & Griffin, Eds., Computer Analysis Of Sequence Data,Part I, Humana Press, New Jersey, (1994); von Heinje, Sequence AnalysisIn Molecular Biology, Academic Press, (1987); and Gribskov & Devereux,Eds., Sequence Analysis Primer, M Stockton Press, New York, (1991).Methods for aligning polynucleotides or polypeptides are codified incomputer programs, including the GCG program package (Devereux et al.,Nuc. Acids Res. 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul et al.,J. Molec. Biol. 215:403 (1990)), and Bestfit program (Wisconsin SequenceAnalysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, 575 Science Drive, Madison, Wis. 53711) whichuses the local homology algorithm of Smith and Waterman (Adv. App.Math., 2:482-489 (1981)). For example, the computer program ALIGN whichemploys the FASTA algorithm can be used, with an affine gap search witha gap open penalty of −12 and a gap extension penalty of −2.

When using any of the sequence alignment programs to determine whether aparticular sequence is, for instance, about 95% identical to a referencesequence, the parameters are set such that the percentage of identity iscalculated over the full length of the reference polynucleotide and thatgaps in identity of up to 5% of the total number of nucleotides in thereference polynucleotide are allowed.

Variant polypeptides can generally be identified by modifying one of thepolypeptide sequences of the invention, and evaluating the properties ofthe modified polypeptide to determine if it is a biological equivalent.A variant is a biological equivalent if it reacts substantially the sameas a polypeptide of the invention in an assay such as animmunohistochemical assay, an enzyme-linked immunosorbent Assay (ELISA),a radioimmunoassay (RIA), immunoenzyme assay or a western blot assay,e.g. has 90-110% of the activity of the original polypeptide. In oneembodiment, the assay is a competition assay wherein the biologicallyequivalent polypeptide is capable of reducing binding of the polypeptideof the invention to a corresponding reactive antigen or antibody byabout 80, 95, 99, or 100%. An antibody that specifically binds acorresponding wild-type polypeptide also specifically binds the variantpolypeptide.

A conservative substitution is one in which an amino acid is substitutedfor another amino acid that has similar properties, such that oneskilled in the art of peptide chemistry would expect the secondarystructure and hydropathic nature of the polypeptide to be substantiallyunchanged. In general, the following groups of amino acids representconservative changes: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr;(2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg,his; and (5) phe, tyr, trp, his.

A polypeptide of the invention can further comprise a signal (or leader)sequence that co-translationally or post-translationally directstransfer of the protein. The polypeptide can also comprise a linker orother sequence for ease of synthesis, purification or identification ofthe polypeptide (e.g., poly-His), or to enhance binding of thepolypeptide to a solid support. For example, a polypeptide can beconjugated to an immunoglobulin Fc region or bovine serum albumin.

A polypeptide can be covalently or non-covalently linked to an aminoacid sequence to which the polypeptide is not normally associated within nature, i.e., a heterologous amino acid sequence. A heterologousamino acid sequence can be from a non-Anaplasma phagocytophilum, anon-Anaplasma platys organism, a synthetic sequence, or an Anaplasmaphagocytophilum sequence or Anaplasma platys sequence not usuallylocated at the carboxy or amino terminus of a polypeptide of theinvention. Additionally, a polypeptide can be covalently ornon-covalently linked to compounds or molecules other than amino acidssuch as indicator reagents. A polypeptide can be covalently ornon-covalently linked to an amino acid spacer, an amino acid linker, asignal sequence, a stop transfer sequence, a transmembrane domain, aprotein purification ligand, or a combination thereof. A polypeptide canalso be linked to a moiety (i.e., a functional group that can be apolypeptide or other compound) that enhances an immune response (e.g.,cytokines such as IL-2), a moiety that facilitates purification (e.g.,affinity tags such as a six-histidine tag, trpE, glutathione, maltosebinding protein), or a moiety that facilitates polypeptide stability(e.g., polyethylene glycol; amino terminus protecting groups such asacetyl, propyl, succinyl, benzyl, benzyloxycarbonyl ort-butyloxycarbonyl; carboxyl terminus protecting groups such as amide,methylamide, and ethylamide). In one embodiment of the invention aprotein purification ligand can be one or more C amino acid residues at,for example, the amino terminus or carboxy terminus of a polypeptide ofthe invention. An amino acid spacer is a sequence of amino acids thatare not associated with a polypeptide of the invention in nature. Anamino acid spacer can comprise about 1, 5, 10, 20, 100, or 1,000 aminoacids.

If desired, a polypeptide of the invention can be part of a fusionprotein, which can also contain other amino acid sequences, such asamino acid linkers, amino acid spacers, signal sequences, TMR stoptransfer sequences, transmembrane domains, as well as ligands useful inprotein purification, such as glutathione-S-transferase, histidine tag,and Staphylococcal protein A, or combinations thereof. Other amino acidsequences can be present at the C or N terminus of a polypeptide of theinvention to form a fusion protein. More than one polypeptide of theinvention can be present in a fusion protein. Fragments of polypeptidesof the invention can be present in a fusion protein of the invention. Afusion protein of the invention can comprise one or more polypeptides ofthe invention, fragments thereof, or combinations thereof.

Polypeptides of the invention can be in a multimeric form. That is, apolypeptide can comprise one or more copies of a polypeptide of theinvention or a combination thereof. A multimeric polypeptide can be amultiple antigen peptide (MAP). See e.g., Tam, J. Immunol. Methods,196:17-32 (1996).

Polypeptides of the invention can comprise an antigenic determinant thatis recognized by an antibody specific for Anaplasma phagocytophilum orAnaplasma platys or both Anaplasma phagocytophilum and Anaplasma platys.The polypeptide can comprise one or more epitopes (i.e., antigenicdeterminants). An epitope can be a linear epitope, sequential epitope ora conformational epitope. Epitopes within a polypeptide of the inventioncan be identified by several methods. See, e.g., U.S. Pat. No.4,554,101; Jameson & Wolf, CABIOS 4:181-186 (1988). For example, apolypeptide of the invention can be isolated and screened. A series ofshort peptides, which together span an entire polypeptide sequence, canbe prepared by proteolytic cleavage. By starting with, for example,30-mer polypeptide fragments, each fragment can be tested for thepresence of epitopes recognized in an ELISA. For example, in an ELISAassay an Anaplasma phagocytophilum polypeptide, such as a 30-merpolypeptide fragment, is attached to a solid support, such as the wellsof a plastic multi-well plate. A population of antibodies are labeled,added to the solid support and allowed to bind to the unlabeled antigen,under conditions where non-specific absorption is blocked, and anyunbound antibody and other proteins are washed away. Antibody binding isdetected by, for example, a reaction that converts a colorless substrateinto a colored reaction product. Progressively smaller and overlappingfragments can then be tested from an identified 30-mer to map theepitope of interest.

A polypeptide of the invention can be produced recombinantly. Apolynucleotide encoding a polypeptide of the invention can be introducedinto a recombinant expression vector, which can be expressed in asuitable expression host cell system using techniques well known in theart. A variety of bacterial, yeast, plant, mammalian, and insectexpression systems are available in the art and any such expressionsystem can be used. Optionally, a polynucleotide encoding a polypeptidecan be translated in a cell-free translation system. A polypeptide canalso be chemically synthesized or obtained from Anaplasma cells.

An immunogenic polypeptide of the invention can comprise an amino acidsequence shown in SEQ ID NOs:1-21 or fragments thereof. An immunogenicpolypeptide can elicit antibodies or other immune responses (e.g.,T-cell responses of the immune system) that recognize epitopes of apolypeptide having SEQ ID NOs:1-21. An immunogenic polypeptide of theinvention can also be a fragment of a polypeptide that has an amino acidsequence shown in SEQ ID NOs:1-21. An immunogenic polypeptide fragmentof the invention can be about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200or more amino acids in length. An immunogenic polypeptide fragment ofthe invention can be about 200, 175, 150, 125, 100, 90, 80, 70, 60, 50,40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, orless amino acids in length.

Anaplasma Polynucleotides

Polynucleotides of the invention contain less than an entire microbialgenome and can be single- or double-stranded nucleic acids. Apolynucleotide can be RNA, DNA, cDNA, genomic DNA, chemicallysynthesized RNA or DNA or combinations thereof. The polynucleotides canbe purified free of other components, such as proteins, lipids and otherpolynucleotides. For example, the polynucleotide can be 50%, 75%, 90%,95%, 96%, 97%, 98%, 99%, or 100% purified. A nucleic acid moleculeexisting among hundreds to millions of other nucleic acid moleculeswithin, for example, cDNA or genomic libraries, or gel slices containinga genomic DNA restriction digest are not to be considered a purifiedpolynucleotide. The polynucleotides of the invention encode thepolypeptides of the invention described above. In one embodiment of theinvention the polynucleotides encode a polypeptide shown in SEQ IDNOs:1-21 or fragments thereof.

Polynucleotides of the invention can consist of less than about 639,450, 300, 225, 147, 138, 135, 105, 96, 60, 54, 51, 45, 30, 20, 10 orless contiguous, naturally occurring (i.e., Aph or Apl polynucleotides)or non-naturally occurring polynucleotides. Polynucleotides of theinvention can consist of greater than about 10, 20, 30, 45, 51, 54, 60,96, 105, 135, 138, 147, 225, 300, 450, 639 or more contiguous, naturallyoccurring (i.e., Aph or Apl polynucleotides) or non-naturally occurringpolynucleotides. The purified polynucleotides can comprise additionalheterologous nucleotides (that is, nucleotides that are not from Aph orApl) and even additional Aph or Apl nucleotides as long as they do notnaturally occur contiguously with the polynucleotides. Polynucleotidesof the invention can comprise other nucleotide sequences, such assequences coding for linkers, signal sequences, TMR stop transfersequences, transmembrane domains, or ligands useful in proteinpurification such as glutathione-S-transferase, histidine tag, andStaphylococcal protein A.

Polynucleotides of the invention can be isolated. An isolatedpolynucleotide is a naturally-occurring polynucleotide that is notimmediately contiguous with one or both of the 5′ and 3′ flankinggenomic sequences that it is naturally associated with. An isolatedpolynucleotide can be, for example, a recombinant DNA molecule of anylength, provided that the nucleic acid sequences naturally foundimmediately flanking the recombinant DNA molecule in anaturally-occurring genome is removed or absent. Isolatedpolynucleotides also include non-naturally occurring nucleic acidmolecules.

Polynucleotides of the invention can also comprise fragments that encodeimmunogenic polypeptides. Polynucleotides of the invention can encodefull-length polypeptides, polypeptide fragments, and variant or fusionpolypeptides.

Degenerate nucleotide sequences encoding polypeptides of the invention,as well as homologous nucleotide sequences that are at least about 80,or about 85, 90, 91, 92, 9394, 95, 96, 97, 98, or 99% identical to thepolynucleotide sequences of the invention and the complements thereofare also polynucleotides of the invention. Percent sequence identity canbe calculated as described in the “Polypeptides” section. Degeneratenucleotide sequences are polynucleotides that encode a polypeptide ofthe invention or fragments thereof, but differ in nucleic acid sequencefrom the wild-type polynucleotide sequence, due to the degeneracy of thegenetic code. Complementary DNA (cDNA) molecules, species homologs, andvariants of polynucleotides of the invention that encode biologicallyfunctional polypeptides also are polynucleotides of the invention.

Polynucleotides of the invention can be obtained from nucleic acidsequences present in, for example, a biological sample, such as blood,serum, saliva, or tissue from an infected individual. Polynucleotidescan also be synthesized in the laboratory, for example, using anautomatic synthesizer. An amplification method such as PCR can be usedto amplify polynucleotides from either genomic DNA or cDNA encoding thepolypeptides.

Polynucleotides of the invention can comprise coding sequences fornaturally occurring polypeptides or can encode altered sequences that donot occur in nature. If desired, polynucleotides can be cloned into anexpression vector comprising expression control elements, including forexample, origins of replication, promoters, enhancers, or otherregulatory elements that drive expression of the polynucleotides of theinvention in host cells. An expression vector can be, for example, aplasmid, such as pBR322, pUC, or ColE1, or an adenovirus vector, such asan adenovirus Type 2 vector or Type 5 vector. Optionally, other vectorscan be used, including but not limited to Sindbis virus, simian virus40, alphavirus vectors, poxvirus vectors, and cytomegalovirus andretroviral vectors, such as murine sarcoma virus, mouse mammary tumorvirus, Moloney murine leukemia virus, and Rous sarcoma virus.Minichromosomes such as MC and MC1, bacteriophages, phagemids, yeastartificial chromosomes, bacterial artificial chromosomes, virusparticles, virus-like particles, cosmids (plasmids into which phagelambda cos sites have been inserted) and replicons (genetic elementsthat are capable of replication under their own control in a cell) canalso be used.

Methods for preparing polynucleotides operably linked to an expressioncontrol sequence and expressing them in a host cell are well-known inthe art. See, e.g., U.S. Pat. No. 4,366,246. A polynucleotide of theinvention is operably linked when it is positioned adjacent to or closeto one or more expression control elements, which direct transcriptionand/or translation of the polynucleotide.

Polynucleotides of the invention can be used, for example, as probes orprimers, for example, PCR primers, to detect the presence of Anaplasmapolynucleotides in a test sample, such as a biological sample. Probesare molecules capable of interacting with a target nucleic acid,typically in a sequence specific manner, for example, throughhybridization. Primers are a subset of probes that can support anenzymatic manipulation and that can hybridize with a target nucleic acidsuch that the enzymatic manipulation occurs. A primer can be made fromany combination of nucleotides or nucleotide derivatives or analogsavailable in the art that do not interfere with the enzymaticmanipulation.

A probe or primer can be about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200or more contiguous nucleotides that encode polypeptides of theinvention.

The hybridization of nucleic acids is well understood in the art anddiscussed herein. Typically a probe can be made from any combination ofnucleotides or nucleotide derivatives or analogs available in the art.The ability of such probes and primers to specifically hybridize toAnaplasma phagocytophilum or Anaplasma platys polynucleotide sequenceswill enable them to be of use in detecting the presence of complementarysequences in a given test sample. Polynucleotide probes and primers ofthe invention can hybridize to complementary sequences in a test samplesuch as a biological sample, including saliva, sputum, blood, plasma,serum, urine, feces, cerebrospinal fluid, amniotic fluid, wound exudate,or tissue. Polynucleotides from the sample can be, for example,subjected to gel electrophoresis or other size separation techniques orcan be immobilized without size separation. The polynucleotide probes orprimers can be labeled. Suitable labels, and methods for labeling probesand primers, are known in the art, and include, for example, radioactivelabels incorporated by nick translation or by kinase, biotin labels,fluorescent labels, chemiluminescent labels, bioluminescent labels,metal chelator labels and enzyme labels. The polynucleotides from thesample are contacted with the probes or primers under hybridizationconditions of suitable stringencies.

Depending on the application, varying conditions of hybridization can beused to achieve varying degrees of selectivity of the probe or primertowards the target sequence. For applications requiring highselectivity, relatively stringent conditions can be used, such as lowsalt and/or high temperature conditions, such as provided by a saltconcentration of from about 0.02 M to about 0.15 M salt at temperaturesof from about 50° C. to about 70° C. For applications requiring lessselectivity, less stringent hybridization conditions can be used. Forexample, salt conditions from about 0.14 M to about 0.9M salt, attemperatures ranging from about 20° C. to about 55° C. The presence of ahybridized complex comprising the probe or primer and a complementarypolynucleotide from the test sample indicates the presence of Apl or anApl polynucleotide sequence in the sample.

Antibodies

Antibodies of the invention are antibody molecules that specificallybind to an Anaplasma phagocytophilum polypeptide or Anaplasma platyspolypeptide or variant polypeptide of the invention or fragment thereof.An antibody of the invention can be specific for an Aph polypeptide orApl polypeptide or a variant polypeptide or a combination thereof, forexample, an antibody specific for one or more of SEQ ID NOs:1-21. Inanother embodiment of the invention an antibody is specific for anAnaplasma phagocytophilum polypeptide (e.g., an antibody specific forSEQ ID NOs:1, 4, 5, 6, 7, 9, 11, 12, 15, 16, 17, 18, or 20). In anotherembodiment of the invention, an antibody is specific for an Anaplasmaplatys polypeptide (e.g., an antibody specific for SEQ ID NOs:8 or 19).In another embodiment of the invention an antibody is specific for bothan Anaplasma phagocytophilum polypeptide and an Anaplasma platyspolypeptide (e.g., an antibody specific for SEQ ID NOs:2, 3, 10, 13, 14,or 21). One of skill in the art can easily determine if an antibody isspecific for an Anaplasma phagocytophilum polypeptide or Anaplasmaplatys polypeptide using assays described herein. An antibody of theinvention can be a polyclonal antibody, a monoclonal antibody, a singlechain antibody (scFv), or an antigen binding fragment of an antibody.Antigen-binding fragments of antibodies are a portion of an intactantibody comprising the antigen binding site or variable region of anintact antibody, wherein the portion is free of the constant heavy chaindomains of the Fc region of the intact antibody. Examples of antigenbinding antibody fragments include Fab, Fab′, Fab′-SH, F(ab′)₂ and F_(v)fragments.

An antibody of the invention can be any antibody class, including forexample, IgG, IgM, IgA, IgD and IgE. An antibody or fragment thereofbinds to an epitope of a polypeptide of the invention. An antibody canbe made in vivo in suitable laboratory animals or in vitro usingrecombinant DNA techniques. Means for preparing and characterizingantibodies are well know in the art. See, e.g., Dean, Methods Mol. Biol.80:23-37 (1998); Dean, Methods Mol. Biol. 32:361-79 (1994); Baileg,Methods Mol. Biol. 32:381-88 (1994); Gullick, Methods Mol. Biol.32:389-99 (1994); Drenckhahn et al. Methods Cell. Biol. 37:7-56 (1993);Morrison, Ann. Rev. Immunol. 10:239-65 (1992); Wright et al. Crit. Rev.Immunol. 12:125-68 (1992). For example, polyclonal antibodies can beproduced by administering a polypeptide of the invention to an animal,such as a human or other primate, mouse, rat, rabbit, guinea pig, goat,pig, dog, cow, sheep, donkey, or horse. Serum from the immunized animalis collected and the antibodies are purified from the plasma by, forexample, precipitation with ammonium sulfate, followed bychromatography, such as affinity chromatography. Techniques forproducing and processing polyclonal antibodies are known in the art.

“Specifically binds” or “specific for” means that a first antigen, e.g.,an Anaplasma phagocytophilum or Anaplasma platys polypeptide, recognizesand binds to an antibody of the invention with greater affinity than toother, non-specific molecules. A non-specific molecule is an antigenthat shares no common epitope with the first antigen. In a preferredembodiment of the invention a non-specific molecule is not derived fromAnaplasma sp. An Anaplasma sp. is any species of the genus Anaplasma.For example, an antibody raised against a first antigen (e.g., apolypeptide) to which it binds more efficiently than to a non-specificantigen can be described as specifically binding to the first antigen.In one embodiment, an antibody or antigen-binding fragment thereofspecifically binds to a polypeptide of SEQ ID NOs:1-21 or fragmentsthereof when it binds with a binding affinity K_(a) of 10⁷ l/mol ormore. Specific binding can be tested using, for example, anenzyme-linked immunosorbant assay (ELISA), a radioimmunoassay (RIA), ora western blot assay using methodology well known in the art.

Antibodies of the invention include antibodies and antigen bindingfragments thereof that (a) compete with a reference antibody for bindingto SEQ ID NOs:1-21 or antigen binding fragments thereof; (b) binds tothe same epitope of SEQ ID NOs:1-21 or antigen binding fragments thereofas a reference antibody; (c) binds to SEQ ID NOs:1-21 or antigen bindingfragments thereof with substantially the same K_(d) as a referenceantibody; and/or (d) binds to SEQ ID NOs:1-21 or fragments thereof withsubstantially the same off rate as a reference antibody, wherein thereference antibody is an antibody or antigen-binding fragment thereofthat specifically binds to a polypeptide of SEQ ID NOs:1-21 or antigenbinding fragments thereof with a binding affinity K_(a) of 10⁷ l/mol ormore.

Additionally, monoclonal antibodies directed against epitopes present ona polypeptide of the invention can also be readily produced. Forexample, normal B cells from a mammal, such as a mouse, which wasimmunized with a polypeptide of the invention can be fused with, forexample, HAT-sensitive mouse myeloma cells to produce hybridomas.Hybridomas producing Anaplasma-specific antibodies can be identifiedusing RIA or ELISA and isolated by cloning in semi-solid agar or bylimiting dilution. Clones producing Anaplasma-specific antibodies areisolated by another round of screening. Monoclonal antibodies can bescreened for specificity using standard techniques, for example, bybinding a polypeptide of the invention to a microtiter plate andmeasuring binding of the monoclonal antibody by an ELISA assay.Techniques for producing and processing monoclonal antibodies are knownin the art. See e.g., Kohler & Milstein, Nature, 256:495 (1975).Particular isotypes of a monoclonal antibody can be prepared directly,by selecting from the initial fusion, or prepared secondarily, from aparental hybridoma secreting a monoclonal antibody of a differentisotype by using a sib selection technique to isolate class-switchvariants. See Steplewski et al., P.N.A.S. U.S.A. 82:8653 1985; Spria etal., J. Immunolog. Meth. 74:307, 1984. Monoclonal antibodies of theinvention can also be recombinant monoclonal antibodies. See, e.g., U.S.Pat. No. 4,474,893; U.S. Pat. No. 4,816,567. Antibodies of the inventioncan also be chemically constructed. See, e.g., U.S. Pat. No. 4,676,980.

Antibodies of the invention can be chimeric (see, e.g., U.S. Pat. No.5,482,856), humanized (see, e.g., Jones et al., Nature 321:522 (1986);Reichmann et al., Nature 332:323 (1988); Presta, Curr. Op. Struct. Biol.2:593 (1992)), caninized, canine, or human antibodies. Human antibodiescan be made by, for example, direct immortilization, phage display,transgenic mice, or a Trimera methodology, see e.g., Reisener et al.,Trends Biotechnol. 16:242-246 (1998).

Antibodies that specifically bind Anaplasma phagocytophilum antigens tothe exclusion of Anaplasma platys antigens (e.g., SEQ ID NOs:1, 4, 5, 6,7, 9, 11, 12, 15, 16, 17, 18, or 20), are particularly useful fordetecting the presence of Anaplasma phagocytophilum antigens in asample, such as a serum, blood, plasma, urine, fecal, tissue, cell, orsaliva sample from an Anaplasma phagocytophilum-infected animal.

Antibodies that specifically bind Anaplasma platys antigens to theexclusion of Anaplasma phagocytophilum antigens (e.g., SEQ ID NOs:8 or19), are particularly useful for detecting the presence of Anaplasmaplatys antigens in a sample, such as a serum, blood, plasma, urine,fecal, tissue, or saliva sample from an Anaplasma platys-infectedanimal.

Antibodies that specifically bind Anaplasma platys and Anaplasmaphagocytophilum antigens (e.g., SEQ ID NOs:2, 3, 10, 13, 14, or 21), areparticularly useful for detecting the presence of Anaplasma platys andAnaplasma phagocytophilum antigens in a sample, such as a serum, blood,plasma, urine, fecal, tissue, or saliva sample from an Anaplasmaplatys-infected or Anaplasma phagocytophilum-infected animal.

An immunoassay for an Anaplasma antigen can utilize one antibody orseveral antibodies. An immunoassay for an Anaplasma antigen can use, forexample, a monoclonal antibody specific for an Anaplasma epitope, acombination of monoclonal antibodies specific for epitopes of oneAnaplasma polypeptide, monoclonal antibodies specific for epitopes ofdifferent Anaplasma polypeptides, polyclonal antibodies specific for thesame Anaplasma antigen, polyclonal antibodies specific for differentAnaplasma antigens, or a combination of monoclonal and polyclonalantibodies. Immunoassay protocols can be based upon, for example,competition, direct reaction, or sandwich type assays using, forexample, labeled antibody. Antibodies of the invention can be labeledwith any type of label known in the art, including, for example,fluorescent, chemiluminescent, radioactive, enzyme, colloidal metal,radioisotope and bioluminescent labels. Antibodies of the invention canspecifically bind Aph antigens only, Apl antigens only, or Apl antigensand Apl antigens.

Antibodies of the invention or fragments thereof can be bound to asupport and used to detect the presence of Apl and/or Aph antigens.Supports include, for example, glass, polystyrene, polypropylene,polyethylene, dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, agaroses and magletite.

Antibodies of the invention can further be used to isolate Apl and/orAph organisms or antigens by immunoaffinity columns. The antibodies canbe affixed to a solid support by, for example, adsorbtion or by covalentlinkage so that the antibodies retain their immunoselective activity.Optionally, spacer groups can be included so that the antigen bindingsite of the antibody remains accessible. The immobilized antibodies canthen be used to bind Anaplasma organisms or Anaplasma antigens from asample, such as a biological sample including saliva, serum, sputum,blood, urine, feces, cerebrospinal fluid, amniotic fluid, wound exudate,or tissue. The bound Anaplasma organisms or Anaplasma antigens arerecovered from the column matrix by, for example, a change in pH.

Antibodies of the invention can also be used in immunolocalizationstudies to analyze the presence and distribution of a polypeptide of theinvention during various cellular events or physiological conditions.Antibodies can also be used to identify molecules involved in passiveimmunization and to identify molecules involved in the biosynthesis ofnon-protein antigens. Identification of such molecules can be useful invaccine development. Antibodies of the invention, including, forexample, monoclonal antibodies and single chain antibodies, can be usedto monitor the course of amelioration of a disease caused by Apl and/orAph. By measuring the increase or decrease of antibodies specific forApl, and/or Aph in a test sample from an animal, it can be determinedwhether a particular therapeutic regiment aimed at ameliorating thedisorder is effective. Antibodies can be detected and/or quantifiedusing for example, direct binding assays such as RIA, ELISA, or westernblot assays.

Methods of Detection

The methods of the invention can be used to detect antibodies orspecific binding fragments thereof specific for Anaplasma antigens, Aplantigens, Aph antigens, Apl polynucleotides, Aph polynucleotides or acombination thereof in a test sample, such as a biological sample, anenvironmental sample, or a laboratory sample. A test sample canpotentially comprise Apl polynucleotides, Aph polynucleotides, Aplpolypeptides, Anaplasma sp. polypeptides, Aph polypeptides, antibodiesspecific for Anaplasma sp., antibodies specific for Apl, and/orantibodies specific for Aph, unrelated polynucleotides, polypeptides,antibodies or antigens, combinations of the above, or none of the above.A biological sample can include, for example, sera, blood, cells,plasma, saliva, urine, feces, or tissue from a mammal such as a horse,cat, dog or human. The test sample can be untreated, precipitated,fractionated, separated, diluted, concentrated, or purified.

In one embodiment methods of the invention comprise contacting one ormore polypeptides of the invention with a test sample under conditionsthat allow polypeptide/antibody complexes, i.e., immunocomplexes, toform. That is, polypeptides of the invention specifically bind toantibodies specific for Apl and/or Aph antigens located in the sample.In one embodiment of the invention one or more polypeptides of theinvention (e.g., SEQ ID NOs:1, 4, 5, 6, 7, 9, 11, 12, 15, 16, 17, 18, 20or fragments thereof) specifically bind to antibodies that are specificfor Aph antigens and do not specifically bind to Apl antigens. Inanother embodiment of the invention one or more polypeptides of theinvention (e.g., SEQ ID NOs:2, 3, 10, 13, 14, 21 or fragments thereof)specifically bind to antibodies that are specific for both Aph and Aplantigens. In one embodiment of the invention one or more polypeptides ofthe invention (e.g., SEQ ID NOs:8, 19 or fragments thereof) specificallybind to antibodies that are specific for Apl antigens and do notspecifically bind to Aph antigens. One of skill in the art is familiarwith assays and conditions that are used to detect antibody/polypeptidecomplex binding. The formation of a complex between polypeptides andanti-Apl and/or anti-Aph antibodies in the sample is detected. Theformation of antibody/polypeptide complexes is an indication thatAnaplasma phagocytophilum polypeptides and/or Anaplasma platyspolypeptides are present in the sample. The lack of detection of thepolypeptide/antibody complexes is an indication that an Anaplasmaphagocytophilum polypeptides and/or Anaplasma platys polypeptides arenot present in the sample.

Antibodies of the invention can be used in a method of the diagnosis ofApl and/or Aph infection by obtaining a test sample from, e.g., a humanor animal suspected of having an Apl and/or Aph infection. The testsample is contacted with antibodies of the invention under conditionsenabling the formation of antibody-antigen complexes (i.e.,immunocomplexes). One of skill in the art is aware of conditions thatenable and are appropriate for formation of antigen/antibody complexes.The amount of antibody-antigen complexes can be determined bymethodology known in the art. A level that is higher than that formed ina control sample indicates an Apl, and/or Aph infection. A controlsample is a sample that does not comprise any Aph and/or Aplpolypeptides or antibodies specific for Aph and/or Apl. In oneembodiment of the invention the control contains no Anaplasma sp.polypeptides or antibodies specific for Anaplasma sp. In one embodimentof the invention an antibody is specific for Aph antigens only. Inanother embodiment of the invention an antibody is specific for both Aphand Apl antigens. In another embodiment of the invention an antibody isspecific for Apl antigens only. Alternatively, a polypeptide of theinvention can be contacted with a test sample. Antibodies specific forApl, and/or Aph in a positive test sample will form antigen-antibodycomplexes under suitable conditions. The amount of antibody-antigencomplexes can be determined by methods known in the art.

In one embodiment of the invention, Anaplasma phagocytophilum and/orAnaplasma platys infection can be detected in a subject. A biologicalsample is obtained from the subject. One or more purified polypeptidescomprising SEQ ID NOs:1-21 or other polypeptides of the invention arecontacted with the biological sample under conditions that allowpolypeptide/antibody complexes to form. The polypeptide/antibodycomplexes are detected. The detection of the polypeptide/antibodycomplexes is an indication that the mammal has an Anaplasmaphagocytophilum and/or Anaplasma platys infection. The lack of detectionof the polypeptide/antibody complexes is an indication that the mammaldoes not have an Anaplasma phagocytophilum infection or an Anaplasmaplatys infection.

Because SEQ ID NOs:2, 3, 10, 13, 14, and 21 are specific for bothanti-Apl and anti-Aph antibodies, the detected infection can be Aphinfection, Apl infection, or both Apl and Aph infection. Because SEQ IDNOs:1, 4, 5, 6, 7, 9, 11, 12, 15, 16, 17, 18, and 20 are specific foranti-Aph antibodies, the detected infection is an Aph infection. BecauseSEQ ID NO:8 and 19 are specific for anti-Apl antibodies, the detectedinfection is an Apl infection. The lack of detection ofpolypeptide/antibody complexes is an indication that the subject doesnot have an Anaplasma phagocytophilum or an Anaplasma platys infection.

In one embodiment of the invention, Apl and/or Aph infection can bedetected in a subject by about 5 days, 6 days, 7 days, 8 days, 9 days,10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days,18 days, 19 days, 20 days, 21 days or more after the subject acquiredthe Apl and/or Apl infection. In one embodiment of the invention, Apland/or Aph infection can be detected in a subject by about 21 days, 20days, 19 days, 18 days, 17 days, 16 days, 15 days, 14 days, 13 days, 12days, 11 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, or lessafter the subject acquired the Apl and/or Apl infection.

In one embodiment of the invention, the polypeptide/antibody complex isdetected when an indicator reagent, such as an enzyme conjugate, whichis bound to the antibody, catalyzes a detectable reaction. Optionally,an indicator reagent comprising a signal generating compound can beapplied to the polypeptide/antibody complex under conditions that allowformation of a polypeptide/antibody/indicator complex. Thepolypeptide/antibody/indicator complex is detected. Optionally, thepolypeptide or antibody can be labeled with an indicator reagent priorto the formation of a polypeptide/antibody complex. The method canoptionally comprise a positive or negative control.

In one embodiment of the invention, one or more antibodies of theinvention are attached to a solid phase or substrate. A test samplepotentially comprising a protein comprising a polypeptide of theinvention is added to the substrate. One or more antibodies thatspecifically bind polypeptides of the invention are added. Theantibodies can be the same antibodies used on the solid phase or can befrom a different source or species and can be linked to an indicatorreagent, such as an enzyme conjugate. Wash steps can be performed priorto each addition. A chromophore or enzyme substrate is added and coloris allowed to develop. The color reaction is stopped and the color canbe quantified using, for example, a spectrophotometer.

In another embodiment of the invention, one or more antibodies of theinvention are attached to a solid phase or substrate. A test samplepotentially comprising a protein comprising a polypeptide of theinvention is added to the substrate. Second anti-species antibodies thatspecifically bind polypeptides of the invention are added. These secondantibodies are from a different species than the solid phase antibodies.Third anti-species antibodies are added that specifically bind thesecond antibodies and that do not specifically bind the solid phaseantibodies are added. The third antibodies can comprise an indicatorreagent such as an enzyme conjugate. Wash steps can be performed priorto each addition. A chromophore or enzyme substrate is added and coloris allowed to develop. The color reaction is stopped and the color canbe quantified using, for example, a spectrophotometer.

Assays of the invention include, but are not limited to those based oncompetition, direct reaction or sandwich-type assays, including, but notlimited to enzyme linked immunosorbent assay (ELISA), western blot, IFA,radioimmunoassay (RIA), hemagglutination (HA), fluorescence polarizationimmunoassay (FPIA), and microtiter plate assays (any assay done in oneor more wells of a microtiter plate). One assay of the inventioncomprises a reversible flow chromatographic binding assay, for example aSNAP® assay. See U.S. Pat. No. 5,726,010.

Assays can use solid phases or substrates or can be performed byimmunoprecipitation or any other methods that do not utilize solidphases. Where a solid phase or substrate is used, one or morepolypeptides of the invention are directly or indirectly attached to asolid support or a substrate such as a microtiter well, magnetic bead,non-magnetic bead, column, matrix, membrane, fibrous mat composed ofsynthetic or natural fibers (e.g., glass or cellulose-based materials orthermoplastic polymers, such as, polyethylene, polypropylene, orpolyester), sintered structure composed of particulate materials (e.g.,glass or various thermoplastic polymers), or cast membrane film composedof nitrocellulose, nylon, polysulfone or the like (generally syntheticin nature). In one embodiment of the invention a substrate is sintered,fine particles of polyethylene, commonly known as porous polyethylene,for example, 10-15 micron porous polyethylene from Chromex Corporation(Albuquerque, N. Mex.). All of these substrate materials can be used insuitable shapes, such as films, sheets, or plates, or they may be coatedonto or bonded or laminated to appropriate inert carriers, such aspaper, glass, plastic films, or fabrics. Suitable methods forimmobilizing peptides on solid phases include ionic, hydrophobic,covalent interactions and the like.

In one type of assay format, one or more polypeptides can be coated on asolid phase or substrate. A test sample suspected of containing ananti-Apl, and/or anti-Aph antibody or antigen-binding fragment thereofis incubated with an indicator reagent comprising a signal generatingcompound conjugated to an antibody or antigen-binding antibody fragmentspecific for Apl and/or Aph for a time and under conditions sufficientto form antigen/antibody complexes of either antibodies of the testsample to the polypeptides of the solid phase or the indicator reagentcompound conjugated to an antibody specific for Apl and/or Aph to thepolypeptides of the solid phase. The reduction in binding of theindicator reagent conjugated to an anti-Apl and/or anti-Aph antibody tothe solid phase can be quantitatively measured. A measurable reductionin the signal compared to the signal generated from a confirmed negativeApl and/or confirmed negative Aph test sample indicates the presence ofanti-Apl and/or anti-Aph antibody in the test sample. This type of assaycan quantitate the amount of anti-Apl and/or anti-Aph antibodies in atest sample.

In another type of assay format, one or more polypeptides of theinvention are coated onto a support or substrate. A polypeptide of theinvention is conjugated to an indicator reagent and added to a testsample. This mixture is applied to the support or substrate. Ifantibodies specific for Apl and/or Aph are present in the test samplethey will bind the one or more polypeptides conjugated to an indicatorreagent and to the one or more polypeptides immobilized on the support.The polypeptide/antibody/indicator complex can then be detected. Thistype of assay can quantitate the amount of anti-Apl, and/or anti-Aphantibodies in a test sample.

In another type of assay format, one or more polypeptides of theinvention are coated onto a support or substrate. The test sample isapplied to the support or substrate and incubated. Unbound componentsfrom the sample are washed away by washing the solid support with a washsolution. If Apl-specific, and/or Aph-specific antibodies are present inthe test sample, they will bind to the polypeptide coated on the solidphase. This polypeptide/antibody complex can be detected using a secondspecies-specific antibody that is conjugated to an indicator reagent.The polypeptide/antibody/anti-species antibody indicator complex canthen be detected. This type of assay can quantitate the amount ofanti-Apl and/or anti-Aph antibodies in a test sample.

The formation of a polypeptide/antibody complex or apolypeptide/antibody/indicator complex can be detected by e.g.,radiometric, colorimetric, fluorometric, size-separation, orprecipitation methods. Optionally, detection of a polypeptide/antibodycomplex is by the addition of a secondary antibody that is coupled to anindicator reagent comprising a signal generating compound. Indicatorreagents comprising signal generating compounds (labels) associated witha polypeptide/antibody complex can be detected using the methodsdescribed above and include chromogenic agents, catalysts such as enzymeconjugates fluorescent compounds such as fluorescein and rhodamine,chemiluminescent compounds such as dioxetanes, acridiniums,phenanthridiniums, ruthenium, and luminol, radioactive elements, directvisual labels, as well as cofactors, inhibitors, magnetic particles, andthe like. Examples of enzyme conjugates include alkaline phosphatase,horseradish peroxidase, beta-galactosidase, and the like. The selectionof a particular label is not critical, but it will be capable ofproducing a signal either by itself or in conjunction with one or moreadditional substances.

Formation of the complex is indicative of the presence of anti-Apland/or anti-Aph antibodies in a test sample. Therefore, the methods ofthe invention can be used to diagnose Apl and/or Aph infection in apatient.

The methods of the invention can also indicate the amount or quantity ofanti-Apl and/or anti-Aph antibodies in a test sample. With manyindicator reagents, such as enzyme conjugates, the amount of antibodypresent is proportional to the signal generated. Depending upon the typeof test sample, it can be diluted with a suitable buffer reagent,concentrated, or contacted with a solid phase without any manipulation.For example, it usually is preferred to test serum or plasma samplesthat previously have been diluted, or concentrated specimens such asurine, in order to determine the presence and/or amount of antibodypresent.

The invention further comprises assay kits (e.g., articles ofmanufacture) for detecting anti-Apl and/or anti-Aph antibodies orantibody fragments, Apl polypeptides, and/or Aph polypeptides in asample. A kit comprises one or more polypeptides of the invention andmeans for determining binding of the polypeptide to anti-Apl antibodiesand/or or anti-Aph antibodies or antigen-binding antibody fragments inthe sample. A kit or article of manufacture can also comprise one ormore antibodies or antigen-binding antibody fragments of the inventionand means for determining binding of the antibodies or antigen-bindingantibody fragments to Apl polypeptides, and/or Aph polypeptides in thesample. A kit can comprise a device containing one or more polypeptidesor antibodies of the invention and instructions for use of the one ormore polypeptides or antibodies for, e.g., the identification of Apland/or Aph infection in a mammal. The kit can also comprise packagingmaterial comprising a label that indicates that the one or morepolypeptides or antibodies of the kit can be used for the identificationof Apl and/or Aph infection. Other components such as buffers, controls,and the like, known to those of ordinary skill in art, can be includedin such test kits. The polypeptides, antibodies, assays, and kits of theinvention are useful, for example, in the diagnosis of individual casesof Apl and/or Aph infection in a patient, as well as epidemiologicalstudies of Apl and/or Aph outbreaks.

Polypeptides and assays of the invention can be combined with otherpolypeptides or assays to detect the presence of Anaplasma along withother organisms. For example, polypeptides and assays of the inventioncan be combined with reagents that detect heartworm and/or Borreliaburgdorferi and/or Ehrlichia canis.

Polynucleotides of the invention can be used to detect the presence ofApl and/or Aph polynucleotides in a sample. The polynucleotides can beused to detect Apl and/or Aph polynucleotides in a sample by a simplehybridization reaction and can also be used in, e.g., polymerase chainreactions (PCR) such as a real-time PCR reaction. Methods andcompositions of the invention can also be used to differentially detectthe presence Aph from other Anaplasma sp., such as Apl.

PCR assays are well described in the art, including, for example, U.S.Pat. No. 4,683,195; U.S. Pat. No. 4,683,202;U.S. Pat. No. 4,965,188.Generally, polynucleotide primers are annealed to denatured strands of atarget nucleic acid. Primer extension products are formed bypolymerization of deoxynucleoside triphosphates by a polymerase. PCRthen involves repetitive cycles of template nucleic acid denaturation,primer annealing and extension of the annealed primers by the action ofa thermostable polymerase. The process results in exponentialamplification of the target Anaplasma sp. nucleic acids in the testsample, which allows for the detection of target polynucleotidesexisting in very low concentrations in a sample.

Real-time PCR assays are based on the detection of a signal, e.g., afluorescent reporter signal. This signal increases in direct proportionto the amount of PCR product in a reaction. Real-time PCR is anyamplification technique that makes it possible to monitor the evolutionof an ongoing amplification reaction. See, Quantitation of DNA/RNA UsingReal-Time PCR Detection, Perkin Elmer Applied Biosystems (1999); PCRProtocols (Academic Press New York, 1989). By recording the amount offluorescence emission at each cycle, it is possible to monitor the PCRreaction during exponential phase where the first significant increasein the amount of PCR product correlates to the initial amount of targettemplate. The higher the starting copy number of the nucleic acidtarget, the sooner a significant increase in fluorescence is observed.

One embodiment of the invention provides a method for detecting and/orquantifying Apl and/or Aph polynucleotides in a test sample. Senseprimers and antisense primers can be added to a test sample underconditions suitable for a polymerase chain reaction. The primershybridize with Apl and/or Aph polynucleotides such that an amplificationproduct is formed if Apl and/or Aph polynucleotides are present in thetest sample. Amplification products are detected and the presence and/orquantity of Apl and/or Aph polynucleotides is determined. Amplificationproducts can be detected with a polynucleotide probe that hybridizes,under conditions suitable for a polymerase chain reaction, with an Apland/or Aph polynucleotide sequence. The amplification product can bequantified by measuring a detection signal from the probe and comparingsaid detection signal to a second probe detection signal from aquantification standard. The quantification standard can be extracted inparallel with the test sample.

One embodiment of the invention provides a method for differentiallydetecting Anaplasma phagocytophilum from Anaplasma platys polypeptidesin a sample. The method comprises:

-   -   (a) contacting one or more antibodies that specifically bind to        a polypeptide consisting of SEQ ID NOs:1, 4, 5, 6, 7, 9, 11, 12,        15, 16, 17, 18, or 20 with a sample under conditions that allow        polypeptide/antibody complexes to form and detecting the        polypeptide/antibody complexes; and    -   (b) contacting one or more antibodies that specifically bind to        a polypeptide consisting of SEQ ID NO:8 or 19 with the sample        under conditions that allow polypeptide/antibody complexes to        form and detecting the polypeptide/antibody complexes.

If the polypeptide/antibody complexes are detected in step (a) and instep (b) then the sample contains Anaplasma phagocytophilum polypeptidesand contains Anaplasma platys polypeptides. If the polypeptide/antibodycomplexes are detected in step (a) and are not detected in step (b) thenthe sample contains Anaplasma phagocytophilum polypeptides and does notcontain Anaplasma platys polypeptides. If the polypeptide/antibodycomplexes are not detected in step (a) and are detected in step (b) thenthe sample contains Anaplasma platys polypeptides and does not containAnaplasma phagocytophilum polypeptides. If the polypeptide complexes arenot detected in step (a) and are not detected in step (b) then thesample does not contain Anaplasma platys polypeptides and does notcontain Anaplasma phagocytophilum polypeptides.

Another embodiment of the invention provides a method of detectingantibodies that specifically bind an Anaplasma platys polypeptide, anAnaplasma phagocytophilum polypeptide, or both an Anaplasma platyspolypeptide and an Anaplasma phagocytophilum polypeptide. The methodcomprises:

-   -   (a) contacting one or more purified polypeptides comprising SEQ        ID NOs:1, 4, 5, 6, 7, 9, 11, 12, 15, 16, 17, 18, or 20 with a        test sample, under conditions that allow polypeptide/antibody        complexes to form and detecting the polypeptide/antibody        complexes; and    -   (b) contacting one or more purified polypeptides comprising SEQ        ID NO:8 or 19, wherein the purified polypeptide with a test        sample, under conditions that allow polypeptide/antibody        complexes to form and detecting the polypeptide/antibody        complexes.

If the polypeptide/antibody complexes are detected in step (a) and instep (b) then the sample contains antibodies the specifically bindAnaplasma phagocytophilum polypeptides and Anaplasma platys polypeptides(that is, antibodies that are capable of specifically binding bothAnaplasma platys and Anaplasma phagocytophilum polypeptides). If thepolypeptide/antibody complexes are detected in step (a) and are notdetected in step (b) then the sample contains antibodies thatspecifically bind Anaplasma phagocytophilum polypeptides and does notcontain antibodies that specifically bind Anaplasma platys polypeptides.If the polypeptide/antibody complexes are not detected in step (a) andare detected in step (b) then the sample contains antibodies thatspecifically bind Anaplasma platys polypeptides and does not containantibodies that specifically bind Anaplasma phagocytophilumpolypeptides. If the polypeptide complexes are not detected in step (a)and are not detected in step (b) then the sample does not containantibodies specific for Anaplasma platys polypeptides and does notcontain antibodies specific for Anaplasma phagocytophilum polypeptides.

Methods of Treatment, Amelioration, or Prevention of a Disease Caused byAph and/or Apl

Polypeptides, polynucleotides, and antibodies of the invention can beused to treat, ameliorate, or prevent a disease caused by Apl and/orAph. For example, an antibody, such as a monoclonal antibody of theinvention or antigen-binding fragments thereof, can be administered toan animal, such as a human or dog. In one embodiment of the invention anantibody or antigen-binding fragment thereof is administered to ananimal in a pharmaceutical composition comprising a pharmaceuticallyacceptable carrier. A pharmaceutical composition comprises atherapeutically effective amount of an antibody or antigen-bindingfragments thereof. A therapeutically effective amount is an amounteffective in alleviating the symptoms of an Apl and/or Aph infection orin reducing the amount of Apl and/or Aph organisms in a subject.

Polypeptides or polynucleotides of the invention can be present in animmunogenic composition and used to elicit an immune response in a host.An immunogenic composition or immunogen is capable of inducing an immuneresponse in an animal. An immunogenic polypeptide or polynucleotidecomposition of the invention is particularly useful in sensitizing animmune system of an animal such that, as one result, an immune responseis produced that ameliorates or prevents the effect of Apl and/or Aphinfection. The elicitation of an immune response in animal model can beuseful to determine, for example, optimal doses or administrationroutes. Elicitation of an immune response can also be used to treat,prevent, or ameliorate a disease or infection caused by Apl and/or Aph.An immune response includes humoral immune responses or cell mediatedimmune responses, or a combination thereof. An immune response can alsocomprise the promotion of a generalized host response, e.g., bypromoting the production of defensins.

One embodiment of the invention provides an immunogen that comprises apolypeptide of the invention and one or more additional regions ormoieties covalently joined to the polypeptide at the carboxyl terminusor amino terminus. Each region or moiety can, for example, enhance theimmune response, facilitate purification of the immunogen, or facilitatepolypeptide stability.

The generation of an antibody titer by an animal against Apl and/or Aphcan be important in protection from infection and clearance ofinfection. Detection and/or quantification of antibody titers afterdelivery of a polypeptide or polynucleotide can be used to identifyepitopes that are particularly effective at eliciting antibody titers.Epitopes responsible for a strong antibody response to Apl and/or Aphcan be identified by eliciting antibodies directed against Apl and/orAph polypeptides of different lengths. Antibodies elicited by aparticular polypeptide epitope can then be tested using, for example, anELISA assay to determine which polypeptides contain epitopes that aremost effective at generating a strong response. Polypeptides or fusionproteins that contain these epitopes or polynucleotides encoding theepitopes can then be constructed and used to elicit a strong antibodyresponse.

A polypeptide, polynucleotide, or antibody of the invention can beadministered to a mammal, such as a mouse, rabbit, guinea pig, macaque,baboon, chimpanzee, human, cow, sheep, pig, horse, dog, cat, or toanimals such as chickens or ducks, to elicit antibodies in vivo.Injection of a polynucleotide has the practical advantages of simplicityof construction and modification. Further, injection of a polynucleotideresults in the synthesis of a polypeptide in the host. Thus, thepolypeptide is presented to the host immune system with nativepost-translational modifications, structure, and conformation. Apolynucleotide can be delivered to a subject as “naked DNA.”

Administration of a polynucleotide, polypeptide, or antibody can be byany means known in the art, including intramuscular, intravenous,intrapulmonary, intramuscular, intradermal, intraperitoneal, orsubcutaneous injection, aerosol, intranasal, infusion pump, suppository,mucosal, topical, and oral, including injection using a biologicalballistic gun (“gene gun”). A polynucleotide, polypeptide, or antibodycan be accompanied by a protein carrier for oral administration. Acombination of administration methods can also be used to elicit animmune response. Antibodies can be administered at a daily dose of about0.5 mg to about 200 mg. In one embodiment of the invention antibodiesare administered at a daily dose of about 20 to about 100 mg.

Pharmaceutically acceptable carriers and diluents and veterinarilyacceptable carries and diluents for therapeutic use are well known inthe art and are described in, for example, Remington's PharmaceuticalSciences, Mack Publishing Co. (A. R. Gennaro ed. (1985)). The carriershould not itself induce the production of antibodies harmful to thehost. Such carriers include, but are not limited to, large, slowlymetabolized, macromolecules, such as proteins, polysaccharides such aslatex functionalized SEPHAROSE®, agarose, cellulose, cellulose beads andthe like, polylactic acids, polyglycolic acids, polymeric amino acidssuch as polyglutamic acid, polylysine, and the like, amino acidcopolymers, peptoids, lipitoids, and inactive, avirulent virus particlesor bacterial cells. Liposomes, hydrogels, cyclodextrins, biodegradablenanocapsules, and bioadhesives can also be used as a carrier for acomposition of the invention.

Pharmaceutically acceptable salts can also be used in compositions ofthe invention, for example, mineral salts such as hydrochlorides,hydrobromides, phosphates, or sulfates, as well as salts of organicacids such as acetates, proprionates, malonates, or benzoates.Especially useful protein substrates are serum albumins, keyhole limpethemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanustoxoid, and other proteins well known to those of skill in the art.Compositions of the invention can also contain liquids or excipients,such as water, saline, phosphate buffered saline, Ringer's solution,Hank's solution, glucose, glycerol, dextrose, malodextrin, ethanol, orthe like, singly or in combination, as well as substances such aswetting agents, emulsifying agents, tonicity adjusting agents,detergent, or pH buffering agents. Additional active agents, such asbacteriocidal agents can also be used.

If desired, co-stimulatory molecules, which improve immunogenpresentation to lymphocytes, such as B7-1 or B7-2, or cytokines such asMIP1α, GM-CSF, IL-2, and IL-12, can be included in a composition of theinvention. Optionally, adjuvants can also be included in a composition.Adjuvants are substances that can be used to nonspecifically augment aspecific immune response. Generally, an adjuvant and a polypeptide ofthe invention are mixed prior to presentation to the immune system, orpresented separately, but are presented into the same site of theanimal. Adjuvants can include, for example, oil adjuvants (e.g. Freund'scomplete and incomplete adjuvants) mineral salts (e.g. Alk(SO₄)₂;AlNa(SO₄)₂, AlNH₄(SO₄), Silica, Alum, Al(OH)₃, and Ca₃(PO₄)₂),polynucleotides (i.e. Poly IC and Poly AU acids), and certain naturalsubstances (e.g. wax D from Mycobacterium tuberculosis, as well assubstances found in Corynebacterium parvum, Bordetella pertussis andmembers of the genus Brucella. Adjuvants which can be used include, butare not limited to MF59-0, aluminum hydroxide,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637), referred to asnor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE), and RIBI, which contains threecomponents extracted from bacteria, monophosphoryl lipid A, trehalosedimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/TWEEN®80 (polysorbate) emulsion.

The compositions of the invention can be formulated into ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, injectable formulations, mouthwashes, dentrifices, andthe like. The percentage of one or more polypeptides, polynucleotides,or antibodies of the invention in such compositions and preparations canvary from 0.1% to 60% of the weight of the unit.

Administration of polypeptides, polynucleotides, or antibodies canelicit an immune response in the animal that lasts for at least 1 week,1 month, 3 months, 6 months, 1 year, or longer. Optionally, an immuneresponse can be maintained in an animal by providing one or more boosterinjections of the polypeptide, polynucleotide, or antibodies at 1 month,3 months, 6 months, 1 year, or more after the primary injection. Ifdesired, co-stimulatory molecules or adjuvants can also be providedbefore, after, or together with the compositions.

A composition of the invention comprising a polypeptide, polynucleotide,antibody, or a combination thereof is administered in a mannercompatible with the particular composition used and in an amount that iseffective to elicit an immune response as detected by, for example, anELISA. A polynucleotide can be injected intramuscularly to a mammal,such as a baboon, chimpanzee, dog, or human, at a dose of 1 ng/kg, 10ng/kg, 100 ng/kg, 1000 ng/kg, 0.001 mg/kg, 0.1 mg/kg, or 0.5 mg/kg. Apolypeptide or antibody can be injected intramuscularly to a mammal at adose of 0.01, 0.05, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 5 or 10 mg/kg.

Polypeptides, polynucleotides, or antibodies, or a combination thereofcan be administered either to an animal that is not infected with Apland/or Aph or can be administered to an Apl and/or Aph-infected animal.An immunologically effective amount or therapeutically effective amountmeans the administration of that amount to an individual, either in asingle dose or as part of series, is effective for treatment,amelioration, or prevention of Apl and or Aph infection. The particulardosages of polynucleotide, polypeptides, or antibodies in a compositionwill depend on many factors including, but not limited to the species,age, gender, concurrent medication, general condition of the mammal towhich the composition is administered, and the mode of administration ofthe composition. An effective amount of the composition of the inventioncan be readily determined using only routine experimentation.

All patents, patent applications, and other scientific or technicalwritings referred to anywhere herein are incorporated by referenceherein in their entirety. The invention illustratively described hereinsuitably can be practiced in the absence of any element or elements,limitation or limitations that are not specifically disclosed herein.Thus, for example, in each instance herein any of the terms“comprising”, “consisting essentially of”, and “consisting of” may bereplaced with either of the other two terms, while retaining theirordinary meanings. The terms and expressions which have been employedare used as terms of description and not of limitation, and there is nointention that in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed byembodiments, optional features, modification and variation of theconcepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the description and theappended claims.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

Examples Example 1 Detection of Anti-Aph and Anti-Apl Antibodies withPolypeptides Derived from the Aph p44 Protein

Polypeptides shown in SEQ ID NOs:12-18 and 10 were coated at 0.5 m/mL onImmulon® 4 microtiter plates in carbonate buffer pH 9.6, overnight. Forall examples, the polypeptide shown in SEQ ID NO:10 has an N at position143, an A at position 145, and an I at position 156.

The plates were washed twice with PetChek® wash buffer. The plates wereblocked with 2% TWEEN® 20 (polysorbate)/2.5% sucrose in 0.1M Tris pH7.6, 2 h and then dried with desiccant overnight. A 1:200 dilution oftest samples in conjugate diluent were added to the plates and incubatedfor 40 minutes. The plates were washed 6 times with PetChek® washbuffer. HRP-conjugated rabbit anti-dog IgG (H+L) (Jackson ImmunoResearchLaboratories, Inc., West Grove, Pa.; Cat. No. 304-035-003), diluted1:2000 in conjugate diluent, was added to the plates and incubated for40 minutes. The plates were washed 6 times with PetChek® wash buffer. 60μl of 3,3′,5,5′-tetramethylbenzidine (“TMB”) was added to the plates andincubated for 10 minutes. 50 μl of Stop solution was added and the A650was determined. Test samples were as follows:

APL_(—)13 DPI: Apl experimentally infected dog 13 days post infectionAPL_(—)78 DPI Apl experimentally infected dog 78 days post infectionAPH: pool of samples from 7 Aph infected dogs from Minnesotaneg: pool of samples from 7 random healthy dogsThe results are shown in Table 1 and FIG. 1.

TABLE 1 Peptide SEQ ID NO: APL_13DPI APL_78DPI APH neg Aph rp44 10 1.130.49 2.62 0.13 Aph p44-1 12 0.29 0.24 1.09 0.28 Aph p44-2 13 1.40 1.573.42 0.22 Aph p44-3 14 1.10 0.51 2.39 0.24 Aph p44-4 15 0.22 0.16 3.080.18 Aph p44-4-1 16 0.20 0.14 2.80 0.11 Aph p44-4-2 17 0.19 0.14 2.380.11 Aph p44-4-3 18 0.23 0.16 2.75 0.15

All 8 tested peptides showed positive reactivity to the pool of serafrom 7 Aph infected dogs from Minnesota. rp44, p44-2, and p44-3 showedcross reactivity to the sera of an experimentally Apl infected dog at 2different time points of infection. Reactivities of p44-1, p44-4,p44-4-1, p44-4-2, and p44-4-3 with the sera of the experimentally Aplinfected dog were near background levels. Therefore, polypeptides p44-1,p44-4, p44-4-1, p44-4-2, and p44-4-3 are not cross-reactive to sera fromApl-infected dogs.

Example 2 Species Specific Detection of Aph or Apl in Field Samples fromEndemic Areas

Polypeptides (Aph p44-4 and Apl p44-4 at 0.5 μg/mL; Aph rp44 at 0.25μg/mL) were coated on Immulon® 4 plates in carbonate buffer pH 9.6,overnight. The plates were washed 2× with PetChek® wash buffer and thenblocked with 2% TWEEN® 20 (polysorbate)/2.5% Sucrose in 0.1M Tris pH 7.6for 2 hours. The plates were dried with desiccant overnight. A 25 μLtest sample was mixed with 50 μL peptide:HRP conjugate (0.5 μg/mL forthe p44-4-Aph (SEQ ID NO:15):HRP conjugate, 1 μg/mL for the p44-4-Apl(SEQ ID NO:19):HRP conjugate, and 3 μg/mL for Aph rp44 conjugate) andincubated on the microtiter plate (incubation time was 1 hour in theexperiment shown in FIG. 2, and 1 hour 45 minutes in the experimentshown in FIG. 3. The plates were washed 6 times with PetChek® washbuffer. 60 μl of TMB was added to the plates and incubated for 10minutes. 50 μl of Stop solution was added and the A650 was determined.The cut off value was determined based on reactivities to 10 negativesamples; cutoff=mean+2×SD (standard deviations).

FIG. 2 demonstrates the results using A. platys positive samples fromdogs living in A. platys endemic areas (HP: Arizona, P: Bahamas). rp44(SEQ ID NO:10) provided positive results for 4 of the 5 “HP” samples,and positive results for 7 of the 7 “P” samples. Aph p44-4 (SEQ IDNO:15) provided positive results for 0 of the 5 “HP” samples, and for 0of the 7 “P” samples. Apl p44-4 (SEQ ID NO:19) provided positive resultsfor 5 of the 5 “HP” samples, and 7 of the 7 “P” samples. Therefore, Aphp44-4 does not cross-react with sera from Apl infected dogs.

FIG. 3 demonstrates the results using A. phagocytophilum positivesamples from dogs living in an Aph endemic area (Minnesota “ME”). rp44(SEQ ID NO:10) provided strong positive results for 21 of the 22 MEsamples. Aph p44-4 (SEQ ID NO:15) provided strong positive results for20 of the 22 ME samples. Apl p44-4 (SEQ ID NO:19) provided negativeresults for 18 of the 22 samples and very weak positive results for 4 ofthe 22 ME samples. Therefore, Apl p44-4 can be considered to notcross-react with sera from Aph infected dogs.

Example 3 Time Course Response in an Apl Experimental Infection Model

Polypeptides (Aph p44-4 and Apl p44-4 at 0.5 μg/mL; Aph rp44 at 0.25μg/mL) were coated on Immulon® 4 microtiter plates in carbonate bufferpH 9.6, overnight. The plates were washed 3 times with PetChek® washbuffer. The plates were blocked with 2% TWEEN® 20 (polysorbate)/2.5%sucrose in 0.1M Tris pH 7.6, 2 h and left to dry overnight. A 25 μL testsample was mixed with 50 μL peptide:HRP conjugate (0.5 μg/mL for Aphp44-4 conjugate, 1 μg/mL for Apl p44-4 conjugate, and 3 μg/mL for Aphrp44 conjugate) and incubated on the microtiter plate for 1 hour. Theplates were washed 6 times with PetChek® wash buffer. 60 μl of TMB wasadded and allowed to incubate for 10 minutes. 50 μl of Stop solution wasadded and the A650 was determined. The results are shown in FIG. 4.

The results demonstrate that SEQ ID NO:19 (Apl p44-4) can be used todetect Apl infections. SEQ ID NO:19 can detect Apl infection at about 10days post-infection. Aph p44-4 (SEQ ID NO:15) shows no cross reactivitywith sera from the Apl infected dogs. Aph rp44 (SEQ ID NO:10) crossreacts with sera from the Apl infected dogs at about 14 days and can beused to detect Apl and Aph infection.

Example 4 Time Course Response in an Aph Experimental Infection Model

Aph p44-4 (SEQ ID NO:15) and Aph rp44 (SEQ ID NO:10) were tested fortheir reactivity with sera from experimentally Aph-infected dogs at aseries of time points. 11 random healthy field dog samples were alsotested. The polypeptides were coated on Immulon® 4 microtiter plates(Aph p44-4 at 0.5 μg/mL; Aph rp44 at 0.25 μg/mL) in carbonate buffer pH9.6, overnight. The plates were washed four times with PetChek® washbuffer. The plates were blocked with 2% TWEEN® 20 (polysorbate)/2.5%Sucrose in 0.1M Tris pH 7.6, for 2 hours and then dried with desiccantovernight. A 25 μL test sample was mixed with 50 μL peptide:HRPconjugate (0.5 μg/mL for the p44-4-Aph (SEQ ID NO:15):HRP conjugate, and3 μg/mL for Aph rp44 conjugate) and added to the plates. The plates wereincubated for 1 hour. The plates were washed 6 times with PetChek® washbuffer. TMB was added to the plates and incubated for 10 minutes. 50 μlof Stop solution was added and the A650 was determined. The results areshown in FIG. 5. Aph p44-4 (SEQ ID NO:15) was able to detect Aphinfection at about 10 to 14 days post-infection. Aph rp44 (SEQ ID NO:10)was able to detect Aph infection at about 10 days post-infection.

Example 5 Detection of Acute Aph Infection

Polypeptides were coated at 0.5 μg/mL or 1.0 μg/mL on Immulon® 4 platesin carbonate buffer pH 9.6, overnight. The plates were washed 4 timeswith PetChek® wash buffer and then blocked with 2% TWEEN® 20(polysorbate)/2.5% sucrose in 0.1M Tris pH 7.6 for 2 hours. The plateswere dried with desiccant overnight. 100 μl of a test serum sample at1:200 dilution in sample diluent was incubated for 45 minutes. Theplates were washed 6 times with PetChek® wash buffer. 100 μl ofHRP-conjugated rabbit anti-dog IgG (H+L) (Jackson ImmunoResearchLaboratories, Inc., West Grove, Pa.; Cat. No. 304-035-003) at 1:2000dilution in sample diluent was added and incubated for 45 minutes. Theplates were washed 6 times with PetChek® wash buffer. 60 μl of TMB wasadded to the plates and incubated for 10 minutes. 50 μl of Stop solutionwas added and the A650 was determined. Test samples were as follows:

Positive (“+”) represents a pool of seven sera from late-stage Aphinfected dogs

Negative (“−”) represents a pool of 7 random healthy dog serum samples

VML8, VML14, VML21 and VML156 represent serum samples from four dogsthat were IFA positive for Aph and had acute clinical signs ofanaplasmosis. The results are shown in FIG. 6. Aph p44-4 reacted withthe 4 sera. Aph p44-1 and Aph p44-2 did not react with the 4 sera, whileAph p44-3 weakly reacted with the 4 sera. Therefore, Aph p44-4 and Aphp44-3 can be used to detect Aph in subjects with acute clinical signs ofanaplasmosis.

Example 6 Performance of Peptide p44-4-v

Aph p44-4-v (SEQ ID NO:20) was tested with the samples listed in Table2.

TABLE 2 Sample Name Sample Composition VML21 dog with acute clinicalsigns of Aph infection, IFA positive for Aph ILS73 dog with acuteclinical signs of Aph infection, IFA positive for Aph, morulae positiveAPH Aph experimental infection, 14 days post infection APL Aplexperimental infection, 13 days post infection +ve pool of samples from7 Aph infected dogs from Minnesota −ve pool of samples from 7 randomhealthy dogs

Polypeptides were coated at 0.5 μg/mL on Immulon® 4 plates in carbonatebuffer pH 9.6, overnight. The plates were washed 2 times with PetChek®wash buffer and then blocked with 2% TWEEN® 20 (polysorbate)/2.5%sucrose in 0.1M Tris pH 7.6 for 2 hours. The plates were dried withdesiccant overnight. The test sample in a 1:200 dilution of conjugatediluent were added to the plates and incubated for 40 minutes. Theplates were washed 6 times with PetChek® wash buffer. HRP conjugatedrabbit anti-dog IgG (H+L) (Jackson ImmunoResearch Laboratories, Inc.,West Grove, Pa.; Cat. No. 304-035-003) in a 1:2000 dilution of conjugatediluent was added to the plates and incubated for 40 minutes. The plateswere washed 6 times with PetChek® wash buffer. 60 μl of TMB was added tothe plates and incubated for 10 minutes. 50 μl of Stop solution wasadded and the A650 was determined. The results are shown in FIG. 7, andrepresent the average of duplicate experiments. p44-4-v providedpositive results for the following samples: VML21; ILS73, APH, and +ve;and negative results for the following samples: APL and −ve. Therefore,Aph p44-4-v can detect Aph infection in acute cases, and at least asearly as 14 days post-infection. Aph p44-4-v is not cross-reactive withsera from Apl infected dogs.

We claim:
 1. A composition comprising one or more purified polypeptides,wherein the one or more polypeptides: (a) consist of an amino acidsequence set forth as SEQ ID NO:13 or 21, or (b) consist of at least 30contiguous amino acids of an amino acid sequence set forth as SEQ IDNO:13; consist of at least 175 contiguous of an amino acid sequence setforth as SEQ ID NO:21, wherein the one or more purified polypeptidesretain an N-terminal C amino acid, or (c) consist of a polypeptidefragment having 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO:2 or SEQID NO:10 over the full length of SEQ ID NO:2 or SEQ ID NO:10, whereinthe sequence of the one or more purified polypeptides is non-naturallyoccurring; or (d) consist of a polypeptide fragment having at leastabout 94% identity to SEQ ID NO:13 or 21 over the full length of SEQ IDNO:13 or 21, wherein the sequence of the one or more purifiedpolypeptides is non-naturally occurring; or (e) comprise an amino acidsequence set forth as SEQ ID NO:13, wherein the one or more polypeptideshave less than 50 contiguous naturally occurring Anaplasma amino acids,wherein the sequence of the one or more purified polypeptides isnon-naturally occurring; wherein, optionally, the one or more purifiedpolypeptides are linked to an indicator reagent, a signal sequence, astop transfer sequence, an amino acid spacer, a transmembrane domain, aprotein purification ligand, a heterologous polypeptide, a moiety thatenhances an immune response, a moiety that facilitates purification, amoiety that facilitates polypeptide stability, one or more additionalpolypeptides comprising any one of SEQ ID NOs:1-21 or a combinationthereof.
 2. The composition of claim 1, further comprising apharmaceutically-acceptable or veterinarily acceptable carrier.
 3. Thecomposition of claim 1, wherein the one or more purified polypeptidesare attached to a solid support.
 4. The composition of claim 1, whereinthe one or more purified polypeptides are chemically synthesized.
 5. Thecomposition of claim 1, further comprising an adjuvant.
 6. A method ofinducing an immune response in a mammal comprising administering animmunologically effective amount of the composition of claim 5 to themammal.
 7. A method of detecting antibodies that specifically bind anAnaplasma platys polypeptide or an Anaplasma phagocytophilum polypeptidein a test sample, comprising: (a) contacting the composition of claim 1with the test sample, under conditions that allow polypeptide/antibodycomplexes to form; (b) detecting the polypeptide/antibody complexes;wherein the detection of the polypeptide/antibody complexes is anindication that antibodies specific for an Anaplasma platys polypeptideor an Anaplasma phagocytophilum polypeptide are present in the testsample.
 8. The method of claim 7, further comprising contacting thecomplexes of step (a) with an indicator reagent prior to the performanceof step (b).
 9. The method of claim 7, wherein the amount of antibodiesin the test sample is determined.
 10. The method of claim 7, wherein theone or more purified polypeptides are attached to a substrate.
 11. Thecomposition of claim 3, wherein the one or more purified polypeptidesare also bound to an antibody that is specific for Anaplasma platys orAnaplasma phagocytophilum.
 12. A device comprising one or more purifiedpolypeptides, wherein the one or more polypeptides consist of an aminoacid sequence set forth as SEQ ID NO:2, 10, 13, 21, or consist of atleast 30 contiguous amino acids of an amino acid sequence set forth asSEQ ID NOs:2 or 13, or consist of at least 175 contiguous amino acids ofan amino acid sequence set forth as SEQ ID NOs:10 or 21, or consist of apolypeptide fragment having at least about 94% identity to any one ofSEQ ID NOs:2, 10, 13, or 21 over the full length of SEQ ID NOs:2, 10,13, or 21; or wherein the one or more polypeptides comprise an aminoacid sequence set forth as SEQ ID NO:2, 10, 13, or 21; wherein the oneor more purified polypeptides are bound to: (a) a solid support; and (b)an antibody that is specific for Anaplasma platys or Anaplasmaphagocytophilum; wherein, optionally, the one or more purifiedpolypeptides are linked to an indicator reagent, a signal sequence, astop transfer sequence, an amino acid spacer, a transmembrane domain, aprotein purification ligand, a heterologous polypeptide, a moiety thatenhances an immune response, a moiety that facilitates purification, amoiety that facilitates polypeptide stability, one or more additionalpolypeptides comprising any one of SEQ ID NOs:1-21 or a combinationthereof.
 13. A composition comprising one or more purified polypeptides,wherein the one or more polypeptides consist of an amino acid sequenceset forth as SEQ ID NO:2, 10, 13, or 21, or consist of at least 30contiguous amino acids of an amino acid sequence set forth as SEQ IDNOs:2 or 13, or consist of at least 175 contiguous amino acids of anamino acid sequence set forth as SEQ ID NOs:10 or 21, or consist of apolypeptide fragment having at least about 94% identity to any one ofSEQ ID NOs:2, 10, 13, or 21 over the full length of SEQ ID NOs:2, 10,13, or 21; or wherein the one or more polypeptides comprise an aminoacid sequence set forth as SEQ ID NO:2, 10, 13, or 21; wherein, the oneor more purified polypeptides are linked to an indicator reagent, asignal sequence, a stop transfer sequence, an amino acid spacer, atransmembrane domain, a protein purification ligand, a heterologouspolypeptide, a moiety that enhances an immune response, a moiety thatfacilitates purification, a moiety that facilitates polypeptidestability, one or more additional polypeptides comprising any one of SEQID NOs:1-21 or a combination thereof.
 14. The composition of claim 13,further comprising a pharmaceutically-acceptable or veterinarilyacceptable carrier.
 15. The composition of claim 13, wherein the one ormore purified polypeptides are attached to a solid support.
 16. Thecomposition of claim 13, wherein the one or more purified polypeptidesare chemically synthesized.
 17. The composition of claim 13, furthercomprising an adjuvant.
 18. The composition of claim 13, wherein the oneor more purified polypeptides are also bound to an antibody that isspecific for Anaplasma platys or Anaplasma phagocytophilum.
 19. A methodof inducing an immune response in a mammal comprising administering animmunologically effective amount of the composition of claim 13 to themammal.
 20. A method of detecting antibodies that specifically bind anAnaplasma platys polypeptide or an Anaplasma phagocytophilum polypeptidein a test sample, comprising: (a) contacting the composition of claim 13with the test sample, under conditions that allow polypeptide/antibodycomplexes to form; (b) detecting the polypeptide/antibody complexes;wherein the detection of the polypeptide/antibody complexes is anindication that antibodies specific for an Anaplasma platys polypeptideor an Anaplasma phagocytophilum polypeptide are present in the testsample.
 21. The method of claim 20, further comprising contacting thecomplexes of step (a) with an indicator reagent prior to the performanceof step (b).
 22. The method of claim 20, wherein the amount ofantibodies in the test sample is determined.
 23. The method of claim 20,wherein the one or more purified polypeptides are attached to asubstrate.